Anton Simeonov Ph.D. National Institute of Health

A review of literature where the authors customize a DNA sequencer to use as an instrument for single-molecule fluorescence detection.

The team from Pacific Biosciences* presents a modified version of their RS II sequencer, which allows the execution of single-molecule fluorescence detection experiments to monitor complex conformational dynamics and reaction events (see Figure 1).

The RS II operates on the principle of zero-mode waveguide (ZMW) detection. ZMWs are arrays of nanosized holes that permit detection of fluorescence emission in extremely small volumes without appreciable background from the bulk medium and thus make possible the scoring of fluorescence events corresponding to individual reaction steps or conformational changes at the single-molecule level. Thus, by immobilizing DNA polymerase onto the ZMW and providing the DNA sequencing template and the four fluorescently labeled deoxynucleotide triphosphates, the system allows for ultrafast DNA sequence determination by scoring the color of each incorporated base as the template is replicated one base at a time. The instrument is able to not only provide very long read lengths but also address templates that have been traditionally very difficult to sequence such as those of high GC content.

Despite this success, the ZMW technology has yet to branch beyond its original application in sequencing. The present report describes a customized ZMW instrument that allows the single molecule–level studies of biological processes at a high throughput using a relatively uncomplicated platform and over relatively long observation periods.

Figure 1. Overview of the customized RS instrument. (A) An SMRT Cell consists of 150,000 ZMWs, of which ~75,000 ZMWs can be simultaneously imaged, allowing multiplexed detection of thousands of single molecules in real time across four spectral channels. (B) Simplified optical schematic of the custom RS instrument: continuous excitation is provided by a 532-nm laser and a 642-nm laser, which are separated into ~75,000 beamlets that illuminate the array of ZMWs on the SMRT Cell that sits on a six-axis stage during data acquisition. The emitted light from the SMRT Cell is collected through the same objective in epifluorescence mode; notch filters in the collection path block transmission of excitation wavelengths. Emitted light is collected on four highspeed complementary metal-oxide-semiconductor (CMOS) cameras. (C) Comparison of workflows for standard sequencing and SMFM experimental mode shows steps for users (square boxes) and instrument (ovals). Modifications made for the SMFM mode reduce time to stage, alignment time, and exposure of components to laser illumination to allow for flexible single-molecule studies with labile reagents. DOE, diffractive optical element; ZMW, zero-move waveguide.

The RS II instrument software and other components were modified to include a new chip alignment algorithm to decrease the amount of photobleaching and allow for a tightly coupled start with the lasers fluid dispense events, reducing variability in the event timings; a different inventory scan routine to enable users to work with custom reagents that may have short half-lives; a customized fluidics protocol to enable flexibility in the single-molecule microscopy studies; and ability to switch between standard sequencing and custom single-molecule study modes.

Pilot experiments with the modified instrument allowed the stepwise recording of the reading of each codon in an RNA template and the corresponding peptide synthesis by the ribosome where immobilized Cy3B-labeled 30S preinitiation complex in the ZMW was used in combination with Black Hole Quencher–50S subunit complex. The two complexes combined and formed the 70S initiation complex, which was characterized by quenched Cy3 signal due to the proximity of Cy3 donor to the Black Hole Quencher.

As translation progressed, the subunits underwent repetitive transitions between rotated and nonrotated states as the codons were read and peptide bonds were made, and this in turn resulted in transient changes in fluorescence, which the instrument was able to record (see Figure 2). Further, by being able to detect four fluorescent channels and follow a large number of individual binding/rearrangement/reaction events simultaneously, the platform allows for the execution of complex conformational dynamics experiments that involve binding and dissociation of multiple interacting ligands. 

Figure 2. Conformational dynamics by FRET on the custom RS. (A) Schematic of experiment showing immobilized Cy3B-labeled 30S preinitiation complex in the ZMW through a biotinylated mRNA and delivery of components. (B) Schematic of the expected signal sequence and example trace of ribosome conformational dynamics during elongation. (C) Rotated state (high intensity and low FRET) lifetimes for each codon, comparable with what we have reported previously (Chen et al., J Am Chem Soc 2012;134:5734–5737; Aitken and Puglisi, Nat Struct Mol Biol 2010;17:793–800). Number of molecules analyzed was 254. Only a portion (~40%) of the entire SMRT Cell was analyzed. (D) Nonrotated (low intensity and high FRET) lifetimes for each codon. All error bars are SEMs. (E) Histogram of ribosomes translating the particular number of codons. Most of the ribosomes translate 12 codons, which was expected from the sequence of the mRNA. The small numbers of additional events beyond the 12th codon shown in the histogram are likely caused by readthrough or statistical errors in the identification of transitions by our analytical method. FRET, fluorescence resonance energy transfer.

*Abstract from PNAS USA 2014, Vol. 111: 664–669

Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.

Anton Simeonov, Ph.D., works at the NIH.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Proceedings of the National Academy of Sciences USA titled "High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence." Authors of the paper are Chen J, Dalal RV, Petrov AN, Tsai A, O'Leary SE, Chapin K, Cheng J, Ewan M, Hsiung PL, Lundquist P, Turner SW, Hsu DR, and Puglisi JD.

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